There are 518 kinases in the human genome, of which at least 30 have been targeted in clinical trials for different diseases. Most of these targeted kinases are tyrosine kinases, and particularly receptor tyrosine kinases, which are targeted by over half of all FDA, approved kinase inhibitors. In many cancers, drugs that target a single kinase often lack efficacy due to the presence of alternate oncogenic signaling pathways that may compensate for the inhibition of the targeted kinase, or mutant versions of the targeted kinase that are unaffected by the candidate compound. Although broadly non-selective kinase inhibitors are highly toxic, it has been shown that selective and simultaneous inhibition of a number of kinases can lead to more effective cancer therapies. Thus, one of the major challenges facing kinase drug discovery is how to efficiently create second generation inhibitors that block multiple selected kinases while avoiding toxicity associated with broad spectrum activity. Although current in vitro biochemical assays are available to measure selectivity of compounds against most of the kinome, these assays are cost-prohibitive to perform on a large scale, and do not adequately replicate the complex cellular environment of the target kinases. Current cell-based assays are usually limited to measuring one kinase per assay well and therefore are not amenable to rapid screening for multipotent inhibitors. Here we propose the creation of a second generation cell-based kinase screening tool that measures the effects of compounds across dozens of kinases in a single well. The ability to simultaneously screen multiple targets will promote the discovery of selectively non-selective compounds that promise more effective therapies. The proposed assay platform takes advantage of Primity's CellCode multiplexing technology which encodes cell populations with unique fluorescent signatures such that multiple cell lines can be combined in a single tube. This technology coupled with the adaptation and optimization of an established, universal, cell-based assay for discovering tyrosine kinase inhibitors enables detailed selectivity measurements and compound screening against the tyrosine kinome at a scale that was previously not possible. PUBLIC HEALTH RELEVANCE: Kinases are implicated in the development and pathogenesis of a wide array of diseases ranging from cancer to neurological and immunological disorders. Here we describe a platform that determines the effects of kinase inhibitors across dozens of targets simultaneously to discover novel drugs with unique patterns of inhibition and to understand potential toxicities of current therapies. This system will improve kinase drug screening and accelerate discovery of novel cancer treatments.